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Bhawani et al

Tropical Journal of Pharmaceutical Research June 2010; 9 (3): 301-313 © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved .

Available online at http://www.tjpr.org Review Article

Thin-Layer Chromatographic Analysis of : A Review

SA Bhawani* 1, O Sulaiman 1, R Hashim 1 and MN Mohamad Ibrahim 2 1Division of Bio-Resource, Paper and Coatings Technology, School of Industrial Technology, 2School of Chemical Sciences, Universiti Sains , 11800, Pulau Pinang, Malaysia

Abstract

Thin layer chromatography has been used for the analysis of natural and synthetic steroids in various environmental materials. This review focuses mainly on analysis in environmental materials such as pharmaceuticals, plant products and other biological specimens. The most widely investigated biological specimens are and blood plasma or serum. Various chromatographic systems useful for the identification; separation and quantification of surfactants are also reported in this review.

Keywords: Steroids; Thin layer chromatography; Environmental materials; Biological specimens

Received: 15 November 2009 Revised accepted: 18 March 2010

*Corresponding author: E-mail: [email protected]

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INTRODUCTION . Steroids, such as , dromostanolone, , are often used Steroids are terpenoid lipids characterized by illegally to increase the performance of the sterane or steroid nucleus: a competitive athletes of almost all age groups. skeleton with four fused rings, generally They are banned in most sports competitions arranged in a 6-6-6-5 fashion. Steroids vary such as the Olympic Games. by the functional groups attached to these rings and the oxidation state of the rings. The Classification of Steroids specificity of their different biological actions is due to the various groups attached to a Steroids have been classified into a number common nucleus. When alcohol groups (OH) of groups by Scott [1] based on their are attached, steroids should properly be functions as follows: (i) and steroid called sterols (e.g., ), whereas alcohols, usually with double bonds; (ii) sex groups (C=O) make them sterones (e.g., - steroids produced mainly in the ). testis (androgens) or ovary (); (iii) adrenocortical hormones - steroids produced Steroids comprise a large group of in the cortex of the adrenal gland; (iv) bile substances that mediate a very varied set of acids - steroids usually bonded to taurine or biological responses. The most widespread in glycine and functioning as emulsion- the body is , an essential stabilizing agents in the intestine; (v) component of membranes and the sapogenins - plant products with a steroid starting point for the synthesis of other bonded to carbohydrates; (vi) cardiac steroids - sex hormones, adrenal cortical glycosides - plant products similar to hormones, and the bile salts. Steroids (e..g., sapogenins and used as heart stimulants; , , and (vii) vitamin D androgens,estrogens and progestagens) have major responsibilities as hormones, ANALYSIS OF STEROIDS controlling , salt balance, and the development and function of the sexual Many procedures used for the quality control organs as well as other biological differences and quality assurance of steroids are based between the sexes. Steroids in the form of on classical methods of analysis. However, bile salts (e.g., salts of cholic and deoxycholic the need for improved precision and accuracy acid and their glycine and taurine conjugates) has led to the increased use of instrumental assist in digestive processes, while another analysis. Thus, the development of fast and steroid is a vitamin (calcitriol) that takes part reliable analytical methods for quality control, in control. Steroids (naturally including the identification of synthesis by- occurring or synthetic) such as products and purity tests, are both important , , gluco- and challenging. Thin-layer chromatography cortisteroids, corticosteroids, squalamine, (TLC) continues to be an important method oestrogens, androgens, are also used for the for qualitative analysis of steroids because of treatment of various diseases such as allergic its inherent advantages - many samples can reactions, arthritis, some malignancies, and be analyzed simultaneously and quickly, and diseases resulting from deficiencies multiple separation techniques and detection or abnormal production. In addition, synthetic procedures can be applied. This review steroids (e.g., ) that mimic the presents the contribution of thin-layer action of are widely used as chromatography in the analysis of steroids oral contraceptive agents. Other synthetic from 1990-2009. It addresses most aspects steroids (e.g., ) are designed to of thin-layer chromatography, including mimic the stimulation of synthesis and detection, separation and quantification of muscle-building action of naturally occurring steroids.

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Thin-layer chromatography of steroids TLC continues to be an important method for the determination of steroids because of its Szepesi and Gazdag wrote a book chapter advantages. Many samples can be analyzed on the TLC of steroids, and it included simultaneously and quickly at relatively low information on sample preparation as well as cost; also, multiple separation techniques and stationary-phase and mobile-phase systems detection procedures can be applied and the useful for the separation of steroidal detection limits are often in the low nanogram pharmaceuticals [2]. The authors also range, and quantitative densitometric provided detailed methods of detection and methods are accurate. Modern approaches in quantification of steroids, and later on, thin-layer chromatography enable analysts to updated their review to include coverage separate and determine steroids in complex through 1994 [3]. Dreassi et al [4] have also mixtures, including various environmental reviewed the application of TLC to steroids in samples. Steroids and their are pharmaceutical analysis while Jain has analyzed by thin-layer chromatography in a provided some information on the analysis of variety of samples such as biological steroid hormones in his review on TLC in samples, plants and pharmaceutical clinical chemistry [5]. formulations. Table 1(a)-(e) shows several thin-layer chromatographic systems designed for the analysis of steroids [6-29].

Table 1(a): Thin-layer chromatographic analysis of steroids

Analyte Stationar Mobile phase Remarks Ref y phase

Cholestrol, RP- Acetronitrile/methanol, Investigation of the retention behavior 6 , HPTLC acetronitrile/water and of 12 sreroids.Mixture of 10g copper pregnanediol, plates methanol/water in different binary sufate and 5 mL o-phosphoric acid etc. mixtures (86%) dissolved in 95 mL methanol. Androgens Silica Cyclohaxane/ethylacetate/ethanol HPTLC separation of anabolic and gestagens (24:16:1) and Androgens. chloroform/benzene/ethanol(36:4:1 Detected by fluorescence after ) in one direction immersion in a 5% sulfuric acid-ethanol ;chloroform/acetone (9:1) and solution for 30 sec and viewed under hexane/dichloromethane/ UV366nm. acetronitrile (4:3:2) in second direction for androgens and gestagens respectively Steroids Silica Chloroform/ethanol/water Detection under 8 (188:12:1) UV.Quantification by radioimmunoassay. Progesterone, Silica Methanol/ethylacetate/chloroform/ Programmed multiple development of 9 , methylenechloride (first inverse analysis of steroids. testosterone gradient program) and Detected under UV 254.Densitometry hydrogen methanol/chloroform (second was used for the quantification. sodium inverse gradient program) salt, etc.

Oxo-steroids Silica gel Chloroform/methanol (97:3) Measurement of 17-oxo steroids in biol. 10 F254 Fluids with TLC and fluorometric scanning detection. Dansylhydrazine was used as a prelabeling reagent. Linearity of fluorescence detection was obtained at 30-1000 ng.

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Table 1(b): Thin-layer chromatographic analysis of steroids ( contd. )

Analyte Stationary Mobile phase Ref phase Hydrocortisone, Benzene/ Use of colour 11 , ethylacetate (1:1) photodocumentation of UV- mesylate, etc. irradiated thin-layer chromatograms for the analysis of steroids.Detected by spraying with a 10% ethanolic solution of sulfuric acid followed by heating at 100 oC for 2 to 4 min Anabolic steroids Silica Chloroform/aceto Analysis of anabolic steroids by 12 ne (9:1) in one high-performance thin-layer direction and chromatography.Detected by cyclohexane/ethy spraying with 10% sulfuric acid in lacetate/methano methanol and heating for 10 min at l (117:78:11) in 95 oC in day light and under UV the opposite 366nm.Further confirmation was direction done by GC-MS

Cortisone, NH 2 F 245s Chloroform/ethan Analysis and separation of steroids 13 hydrocortisone, ol/formic acid on NH 2 layers. , (50:10:10), , Chloroform/meth ,, (95:5), , Chloroform/1- testosterone, propanol/formica testosterone cid (50:10:5) propionate, prednisolone, pegnandiol and triol, progeterone and Reichstein’s S

Cortisole, , NH 2 F 245s Chloroform/ethan Separation and detection of 14 testosterone and ol (95:5) steroids. Plates were heated to progesterone approx. 170 oC for 12 min for fluorescence development. Flourescence can be increased two fold by dipping plates into a mixture of hexane- paraffin (2:1 v/v) Steroids Silica 1.Hexane/ethylac Silver nitrate impregnated silica 15 impregnated etate (3:1,2:1); layers were used for the separation with silver 2.hexane/ether of steroids. nitrate (10:1,5:1); 3.hexane; 4.hexane/tolune (10:1) Allylestrenol, Silica gel Cyclohexane/but One- and two- dimensional HPTLC, 16 , ylacetate/chlorof TLC and personal OPLC analysis ethynodiol, etc. orm (86:7:7); of steroids. toluene/ethylacet ate/chloroform (5:1:4)

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Table 1(c): Thin-layer chromatographic analysis of steroids ( contd. )

Analyte Stationary Mobile phase Remarks Ref phase Chenodeoxycho Silica gel 1. methanol–0.3% sodium Separation of the 17 lic acid and RP-18F 254S phosphate buffer (pH, 7.5). unconjugates and conjugates deoxycholic acid and silica First development. of gel 60F 254 (A, 80:20, v/v) (B, 70:30, chenodeoxycholic acid and v/v) (C, 65:35, v/v) deoxycholic acid by two- 2. n-hexane–ethyl – dimensional reversed-phase ,First thin-layer chromatography development (A, 72:18:10, with methyl β –cyclodextrin. A v/v/v) high degree of separation of 3. methanol–0.3% sodium individual bile acids in each phosphate buffer (pH, 7.5) homologous series was containing 5 m M Me-b-CD achieved on a RP–HPTLC (A, 80:20, v/v) (B, 70:30, plate by developing with v/v) (C, 65:35, v/v),Second aqueous methanol in the first development dimension and the same 4.acetic acid–methanol– solvent system containing water Me- β -CD in the second (A, 60:20:20, v/v/v). dimension. Second development Steroids Silica gel n-hexane/ether/acetic acid Evaluation of a new type of 18 (65:35:1) and only n- radiodetector designed for hexane for the second digital autoradiography of development TLC plates for the detection of steroids. Quantification by densitometry after dipping for 30s in 3% sulfuric acid,drying at 60 oC for 15 min and heating at 160 oC for 15 min. Double Silica gel Methanol/water/0.5 moL - Separation of a series of a 19 1 conjugates of RP-18F 254S tetra-n-butylammonium polar, ionic and hydrophilic bile acids and silica phosphate double conjugates of bile gel 60F 254 (A,90:10:5; B,802:0:5; acids amidated at the C-24 C,75:25:5), for first carboxyl group with the development and glycine or taurine and Methanol/water/0.5 moL - sulfonated or glucosylated at 1tetra-n-butylammonium hydroxyl groups in the 5 β- phosphate containing 5mM steroid nucleus. Me- β-CD (A,90:10:5; B,802:0:5; C,75:25:5), for second development Β-sitosterol, Silanized Methanol/water (1:1) Preparative TLC of steroids 20 , silica gel from Harrisonia abyssinica . , Detected under UV. etc.

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Table 1(d): Thin-layer chromatographic analysis of steroids ( contd. )

Analyte Stationary Mobile Remarks Ref phase phase , 13 β-ethyl- Determination of levonorgestrel 17 β-hydroxy-18,19-dinor- Silica gel Toluene/2- in release media 17 α-pregn- 60F 254 propanol of an in-situ-forming - 21 4-en-20-yn-3-one (90:10) delivery system based on poly(D,L lactide-co-glycolide) and N-methyl-2-pyrrolidone. Densitometric detection and quantification were performed at λ = 250 nm. , Methanol/wat Lipophilicity of selected steroids , Silica RP- er and was determined by RP- testosterone, etc. 18W acetonitrile/w HPTLC.Lipophilicity values 22 ater(in were estimated by different computational methods. compositions) Detected by spraying with sulfuric acid/methanol(1:9) and heating at 120 oC for 15 min. Cortisol and cortisone Thin-layer Silica gel Phosphate chromatographic 60 buffer competitive protein- 23 binding assay for cortisol and cortisone. Specific and rapid detn. of free cortisol and cortisone in human urine. Detected under UV light. Levonorgestrel and Simultaneous detn. of steroidal ethinyloestradiol Silica gel Hexane- hormones levonorgestrel and chloroform- ethinyloestradiol both in bulk 24 methanol drug and in low-dosage oral (1.0:3.0:0.25) contraceptives. Densitometric anal. of the was carried out in the reflectance mode at 225 nm by using a computer controlled densitometric scanner. Ergosterol, stigmasterol, Improved detection of steroids. dihydrocholestrol, 4- Silica gel Methanol/dich Detected by spraying twice with 25 cholesten-3one, loromethane 10 % phosphomolybdic acid in cholecalciferol and (1:9) methanol followed by heating cholesterol acetate Sterols Silica gel Petroleum Semiautomated band-wise 26 ether/diethyl sample application, and ether/glacial automated visible mode acetic acid densitometry was developed (80:20:1) for the determination of the steroids.

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Table 1(e): Thin-layer chromatographic analysis of steroids ( contd .)

Analyte Stationary Mobile phase Remarks Ref phase Progesterone, Silica gel 60 Chloroform/aceto Simultaneous sepn. of 27 acetate, F254 ne eleven steroid hormones acetate, and synthetic anabolics. The 17-β-estradiol, 19- investigated steroids were nortestosterone, successfully visualized , under UV light (254 nm), and , 4- after spraying with an chloro-δ-1-Me ethanolic soln. of p- testosterone, toluenesulfonic acid. acetate, 6- β - hydroxymethandienon e and Ethinyl estradiol, Silica gel Cyclohexanone/e Optimization and 28 , thylacetate/chloro comparison of the acidic Nandrolone, etc. form (1:1:1) visualization of steroids separated by OPLC.Detected with sulfuric acid(under UV 366nm) at three different concentrations, phosphomolybdic acid (in white light) and phosphoric acid (under UV 366nm). Evaluation by videodensitometry

Estradiol, Diol F 254s Chloroform The densitometric detection 29 hydrocortisone, of these compounds with testosterone and and without the use of cholesterol sulfuric acid solutions as visualizing reagents was compared. Comparison and characterization of chromatographic spots of examined compds. on the basis of resolution (RS), separation factor ( α), constance of the pair separation (R α F), and ∆RF values were discussed.

The 5 were analysed by phase composition are very important for the thin-layer chromatography using optimum analysis of androstane isomers by thin-layer mobile phases. The choice of proper mobile chromatography (TLC). In the 1st step, the phase and the optimization of the mobile proper solvent system was found to be the

Trop J Pharm Res, June 2010; 9 (3): 307 Bhawani et al mixture of chloroform, acetone, and solasodine glycosides separated by silica gel petroleum ether chosen from 7 elution TLC were transferred to a polyvinylidene systems. In the second step, the composition difluoride membrane. The membrane was of the mobile phase was optimized by treated with sodium periodate solution and "simplex" and "prisma" methods. The the with bovine serum albumin (BSA), optimum TLC system can be applied for the resulting in a solasodine glycoside-BSA separation of androstane isomers from real conjugate. Individual spots were stained by samples such as drug formulation, biological monoclonal antibody against solamargine. and natural resources [30]. Separation of a The newly established immunostaining method can be extended to the analysis of large number of ecdysteroids was the distribution of solasodine glycosides in investigated with eleven mobile phases and the plant extract [34]. Folin-Ciocalteu's three stationary phases. Only the use of four reagent along with three new solvent systems mobile phases on three stationary phases was used for the study of 9 anabolic steroids enabled the separation of all the ecdysteroids prohibited in sports, namely testosterone from each other in at least one system. The undecanoate, methyltestosterone, TLC behaviour of ecdysteroids containing methandienone, testosterone, testosterone different numbers of hydroxyl groups, side- propionate, nandrolone phenylpropionate, chain variations, and extra double bonds, and ethylsterenol, oxygmetholone, and stanozolol of positional isomers and stereoisomers, was [35]. reported and interpreted [31]. A rapid, selective and precise stability A comparative study has been performed on indicating high performance thin layer the thin-layer chromatographic detection of chromatography method was developed and different corticosteroids. In this study, twelve validated for the estimation of oestradiol different mixtures of organic solvents were (ESD) in bulk and pharmaceutical dosage compared to assess their efficiency as mobile forms [36]. ESD is widely used in post- phases for the separation of eighteen climacteric replacement therapy. The glucocorticosteroids along with four different developed method employed silica gel 60F 254 spray reagents. Optical evaluation of the as the stationary phase and chloroform– plates revealed that the combination of acetone–isopropyl alcohol–glacial acetic acid choice for optimum separation and detection (9:1:0.4:0.1) as mobile phase. The dense and was chloroform-methanol (92:8), or compact spot of the drug occurred at an Rf chloroform-acetone (90:10) as mobile phase value of 0.40 ± 0.02. Spectrodensitometric and a mixture of 2,4-dihydroxybenzaldehyde, scanning-integration was performed on a sulfuric acid, and acetic acid as spray reagent Camag system at a wavelength of 286 nm. [32]. Chromatographic study of 36 estradiol The polynomial regression data for the and estrone was conducted on silica and RP- calibration plots exhibited good linear 18 silica with non-aqueous and aqueous- relationship ( r = 0.9947) over a concentration organic mobile phases. The slopes of the range of 1 – 8 µg. Recovery studies were linear relationship between RM and the also performed at three experimental levels. volume fractions of the polar organic The recovery data revealed that the relative components of the binary eluents were also standard deviation (RSD) for intra-day and calculated [33]. inter-day analysis was found to be 1.27 and 1.75 %, respectively. The intentional acidic A simple thin-layer chromatography degradation of oestradiol gave two products. immunostaining method using monoclonal In the presence of an acid, protonation of the antibody against solamargine was developed 17-β-hydroxyl group occurred, followed by for the determination of solasodine the loss of a water molecule. This would glycosides [34]. In this method, the further result in elimination of a proton at C-

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16 position leading to the formation of a supplements, DS-1 was claimed to contain double bond at C16–17 position (degradation 20E of 100 % pharmaceutical quality with a product I). Furthermore this product could serving portion of 1 capsule, i.e., 300 mg of undergo a possible rearrangement to give a 20E per serving. Dietary supplement, DS-2, double bond at C15–16 position (degradation in addition to other plant extracts and product II). Thus, the two spots additives, contained Ajuga turkestanica and corresponding to the degraded components Rhaponticum carthamoides root extract obtained after acidic degradation, can be standardized to contain 37.5 mg of 20E per attributed to these two alkenes (degradation serving with a serving portion of two capsules products I and II) with not much significant (18.75 mg/capsule). The analysis showed difference in the Rf values (0.52 ± 0.01 ( n = DS-1 and DS-2 to contain 95.1 and 17.28 mg 6) and 0.58 ± 0.01 ( n = 6)). These of 20E per capsule, respectively. compounds, being more non-polar in nature, have R f values higher than the pure drug. The quantitative analysis of (24b- The products may also undergo racemization Ethylcholesta-5, 22E,25-triene-3b-ol) in in acidic conditions. Agnimantha ( Clerodendrum phlomidis Linn) was performed on silica gel 60F 254 plates with The determination of 20-Hydroxyecdysone in chloroform-methanol (98.5:1.5) as the mobile Sida rhombifolia L. and dietary supplements phase [38]. The method employed automated was performed by a simple HPTLC method bandwise sample application, and automated [37]. The developed method employed glass visible mode densitometry for the TLC plates coated with silica gel 60F 254 as determination of 24b-ethylcholesta-5,22E,25- stationary phase and chloroform:methanol triene-3b-ol (ECTO) in the aerial part of (8:2) as developing solvent system. The Clerodendrum phlomidis . The effect of developed method was used to quantitate 20- extracting solvents was studied with respect hydroxyecdysone in methanol extract of the to the content of ECTO in C. phlomidis . whole plant material of Sida rhombifolia and Various extracts, namely, n-hexane, was also successfully applied for the chloroform, ethyl acetate, methanol and quantitative evaluation of dietary ethanol were chromatographed to evaluate supplements. The densitometric evaluation of extraction efficiency as well as interferences 20-hydroxyecdysone (20E) was performed at due to co-eluted compounds. All the sample 250 nm in reflectance/absorbance mode. tracks were scanned at 254 and 366 nm High performance thin-layer chromatography wavelength, in addition to derivatization with (HPTLC) fingerprint of six different Sida anisaldehyde reagent. It is clearly evident species with 20E was obtained and that no interfering compound was eluted in significant amounts of 20E were seen in S. the sample tracks to affect the quantitation of rhombifolia . However, on derivatization with the targeted marker, ECTO. Ethylacetate was anisaldehyde-sulfuric acid reagent, each of found to be the most suitable and exhaustive the species exhibited unique identity in solvent for sample preparation. A precise and fingerprint that can be used in distinguishing accurate quantification was performed in the them. In all the samples, 20E showed good linear working concentration range of 150 – 2 separation with an R f value of 0.37 ± 0.01. 400 ng/band with good correlation (r = The purity of 20E in sample bands was 0.996). This method was also validated for ascertained by comparison of spectral scans peak purities, precision, robustness, limits of and UV maximum (250 nm) with the detection (LOD) and quantitation (LOQ), etc, standard. The five-point calibration curve was as per ICH guidelines. plotted in the linearity range of 200 – 1,000 ng spot -1 of 20E. The two dietary The lipophilicity of some dehydroepian- supplements were also analyzed using the drosterone derivates was evaluated by RP-18 proposed method. Among the two dietary HPTLC chromatography [39]. Lipophilicity is

Trop J Pharm Res, June 2010; 9 (3): 309 Bhawani et al one of the inherent properties of chemical be considered as a homologous series. 0 compounds, affecting their biological activity. Finally, R M values proved to be a reliable Lipophilicity plays a determinant role in the alternative for lipophilicity expression as well transport of compounds through a biological as activity, and can be used for further system and it may also influence the studies of the compounds’ quantitative formation of a complex between a compound structure-activity relationships. and a receptor or a biomacromolecule at the site of action. The chromatographic behavior Steroids in biological samples (urine, etc.) are of DHEA derivates - 17 α-substituted-3β, 17 β also determined by thin-layer -dihydroxy-16-oximino derivatives of 5- chromatography. In one attempt [40], cortisol androstene was studied on a C-18 bonded in urine samples of guinea pig was phase with two aqueous eluents, acetone- determined by HPTLC, HPLC and TLC-RIA, water and dioxane-water. For lipophilicity respectively, and the results obtained by the determination, each experiment was run in three methods were compared. HPTLC and triplicate. For subsequent calculations, mean TLC-RIA was performed on NH 2 F 254 and RM values were used and were calculated as silica gel 60 F 254 , respectively, with the in Eq 1 solvent system consisting of chloroform/methanol/water (90:6:0.5). In the RM = log (1/R f -1) ……………..…………. (1) case of HPLC, C 18 -reversed phase was used with water/methanol (1:1) as the mobile The calculated RM values, with different phase. Following intramuscular concentrations of the organic solvent, administration of 25mg cortisol, cortisol 0 were used for the calculation of R M values. increased from about 10-30µg/day The calculated RM values were to 400-500 µg/day (i.e., HPTLC: 531 µg/day; extrapolated to 0 % of organic modifier HPLC: 493 µg/day; and TLC-RIA: 394 0 concentration (R M) using Eq 2. µg/day). Similarly, treatment of animals with 20 IU adrenocorticotropic hormone (ACTH) 0 RM = R M -S. ф. ………………………..…… (2) resulted in augmented cortisol excretion, with mean values of 294 (HPTLC), 256 (HPLC) where ф is the volume fraction of the organic and 143 µg/day (TLC-RIA), respectively. The solvent in the mobile phase and S is the cortisol amounts measured by the HPTLC change in RM caused by unit change of and HPLC agreed, but the amounts organic modifier concentration in the mobile measured by the TLC-RIA were significantly 0 phase. The R M (intercept) is an extrapolated lower. value obtained at ф = 0% (modifier) and represents the most applied chromatographic On the other hand, a simple, inexpensive and lipophilic parameter. The linear relationship reliable method was developed for the (Eq 2) was obtained over all of the determination of cortisol in plasma and urine investigated concentration ranges for all the of guinea pig by thin-layer chromatography studied substances in acetone-water and and fluorescence derivatization with dioxane-water mobile phases. The calculated isonicoitinic acid hydrazine [41]. The 0 R M (intercept) values are different for each developed method employed silica gel 60 compound and depend only on chemical F254 as stationary phase and structures (i.e., the substituent in the 17 α- chloroform/methanol (9:1, v/v) as mobile 0 position). Although the R M values depend on phase. After development, the plates were the type of organic modifier, there was linear dipped into isonicoitinic acid hydrazine (INH) 0 correlation among R M for acetone and as reagent (3g INH, 5g trichloroacetic acid, and well as for dioxane. The significant correlation 300mL). The fluorescence of the cortisol 0 between the R M values and S-slopes hydrazone was further increased by dipping indicate that investigated compounds could the plates into chloroform-liquid paraffin (9:1).

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The fluorescence was measured unknowns has to be done with more specific densitometrically (excitation 366nm; cut-off techniques, such as infra-red spectroscopy filter: >460nm). The fluorescence intensity and MS detection. was linearly dependent on the amount of cortisol between 1ng - 200ng. The ACKNOWLEDGEMENT coefficients of variation ranged between 6.3 (1ng) and 1.4 % (200 ng). The sensitivity of The first-named author is grateful to Universiti this method (< 1 ng) enables the Sains Malaysia, Malaysia for providing measurement of cortisol in the plasma and postdoctoral fellowship. urine of saline-, ACTH- or cortisol-treated guinea-pigs. Thin-layer chromatography and REFERENCES fluorescence derivatization with isonicoitinic acid hydrazine was also used for the 1.Scott RM .Clinical Analysis by Thin-Layer determination of cortisol and cortisone in Chromatography Techniques. Ann Arbor- human morning and overnight urine [42]. Humphrey Science Publishers, Ann Arbor, MI. Free cortisol and cortisone were also 1969. 2. Szepesi G, Gazdag M. Steroids. In Handbook of Thin measured by thin-layer chromatographic Layer Chromatography,Sherma J, Fried B competitive protein-binding assay in the urine (Eds.) Marcel Dekker, New York, 1991, p. 907. of male individuals who abstained from water 3. Szepesi G, Gazdag M. Steroids. In Handbook of Thin- intake for 2 h or who ingested 1 L of water Layer Chromatography. Sherma J, Fried B (Eds.) Marcel Dekker, New York, 1996, p. 971. [23]. In this study, silica gel 60 F 254 plates and 4. Dreassi E, Ceramelli G, Corti P. Thin-layer acetone–toluene (1:1) were used for the chromatography in pharmaceutical chromatography. Chicken serum was used analysis. In Practical Thin-Layer as the source of corticosteroid binding Chromatography—A Multidisciplinary Approach. Fried B., Sherma J. (Eds.) CRC globulin, because it binds cortisol and Press, Boca Raton, FL, 1996, p. 231. cortisone with a similar high affinity. 5. Jain R. Thin-layer chromatography in clinical chemistry. In Practical Thin-Layer Chromatography—A Multidisciplinary CONCLUSION Approach. Fried B., Sherma J. (Eds.). CRC Press, Bxoca, Raton, FL, 1996, p. 131. Thin layer chromatography (TLC) is a globally 6. Lamparczyk H , Ochocka RJ, Zarzycki P, Zielinski JP. accepted practical solution to characterize Separation of steroids by reversed-phase HPTLC using various binary mobile phases. J. raw herbs, active constituent-enriched Planar Chromatogr., 1990; 3: 34-37. extracts and their formulations. Standardized 7. De Brabander HF,Van Hoof J. HPTLC analysis of TLC procedures can be used effectively for residues of anabolics in meat and fat. screening analysis as well as quality J. Planar Chromatogr., 1990; 3: 236-242. 8. Feuske M, Schonbeiter H. Thin-layer chromatography evaluation of a plant or its derived herbal on silica coated sheet as an adjunct products. Owing to the simplicity and to radioimmunoassay of steroids. J. efficiency of TLC, specific and rapid Chromatogr., 1991; 563: 178- 183. determination of various steroids in humans 9. Matyska M, Siouffi AM, Soczewinski E. Programmed multiple development (PMD) analysis of and various other animals can be carried out. steroids by planar chromatography with a new The procedure can be employed for the modification of the routine analysis of steroids in pharmaceutical horizontal sandwich chamber. J. Planar Chromatogr., formulations and in bulk drug preparations as 1991; 4: 255-257. 10. Zdena T. Thin-layer chromatography of urinary 17- well as for the quality assurance of related oxosteroids using extracts and market samples. Interest in TLC dansylhydrazine as a prelabeling reagent. J. has increased with improvements in TLC Chromatogr. Biomed. Appl., 1991; 570: 396- instrumentation and methods, and especially 398. 11. Vegh Z. True color photodocumentation of UV- in the last few years, with the development of irradiated thin layer chromatograms. new MS methods for detection. If standard J. Planar Chromatogr., 1993; 6: 341-345. compounds are not available, identification of

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